The Spore Differentiation Pathway in the Enteric Pathogen Clostridium difficile

Abstract

Endosporulation is an ancient bacterial developmental program that culminates with the differentiation of a highly resistant endospore. In the model organism Bacillus subtilis, gene expression in the forespore and in the mother cell, the two cells that participate in endospore development, is governed by cell type-specific RNA polymerase sigma subunits. σF in the forespore, and σE in the mother cell control early stages of development and are replaced, at later stages, by σG and σK, respectively. Starting with σF, the activation of the sigma factors is sequential, requires the preceding factor, and involves cell-cell signaling pathways that operate at key morphological stages. Here, we have studied the function and regulation of the sporulation sigma factors in the intestinal pathogen Clostridium difficile, an obligate anaerobe in which the endospores are central to the infectious cycle. The morphological characterization of mutants for the sporulation sigma factors, in parallel with use of a fluorescence reporter for single cell analysis of gene expression, unraveled important deviations from the B. subtilis paradigm. While the main periods of activity of the sigma factors are conserved, we show that the activity of σE is partially independent of σF, that σG activity is not dependent on σE, and that the activity of σK does not require σG. We also show that σK is not strictly required for heat resistant spore formation. In all, our results indicate reduced temporal segregation between the activities of the early and late sigma factors, and reduced requirement for the σF-to-σE, σE-to-σG, and σG-to-σK cell-cell signaling pathways. Nevertheless, our results support the view that the top level of the endosporulation network is conserved in evolution, with the sigma factors acting as the key regulators of the pathway, established some 2.5 billion years ago upon its emergence at the base of the Firmicutes Phylum.

The Clostridium Sporulation Programs: Diversity and Preservation of Endospore Differentiation

“…As expected, the C. difficile sigF mutant was unable to form any heat-resistant spores. TEM image analysis revealed that the sigF disruption mutant had its sporulation blocked at asymmetric division and was unable to progress further in spore morphogenesis, which is similar to the observations of the B. subtilis sigF mutant (25,91,94) (Fig. 5).…”

“…The sigF gene was also disrupted in the important pathogen C. difficile (generating sigF disruption mutant strain AHCD533) (94). As expected, the C. difficile sigF mutant was unable to form any heat-resistant spores.…”

“…However, a more electron-translucent region with some electron-dense layers was observed in some of the C. difficile sigF mutant cells, which is in contrast to the B. subtilis sigF mutant. Additionally, DNA staining showed that the DNA was strongly localized in the forespore in the C. difficile sigF mutant (25,94). Using transcriptional SNAP Cd fusions to CD2470 (the ortholog of the B. subtilis gpr gene), which is predicted to be under the control of F , Pereira et al demonstrated that F is active exclusively in the forespore despite being transcribed in both the mother cell and forespore compartments (94).…”

Clostridium difficile Infection: Pathogenesis, Diagnosis and Treatment

“…The mechanism of regulation of all these factors is not known and is currently under study. However, Pereira et al observed that as in B. subtilis, the activity of factors F and G in C. difficile is focused on forespores and that of factors E and K on the mother cell (Figure 2) [23].…”

Esporas de Clostridium difficile y su relevancia en la persistencia y transmisión de la infección

“…En C. perfringens, σ K es producido en células en la fase pre-divisional y se encuentra involucrado en la producción de enterotoxina 105 . En cuanto a C. difficile, un estudio mostró que en relación a los roles de los distintos factores sigma involucrados en esporulación, C. difficile parece ser cercano al organismo modelo, B. subtilis, más que a otras especies del género Clostridium 106 .…”